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Research on affect in digital learning environments focused almost exclusively on positive emotions. Nevertheless, understanding negative emotions experienced in these environments is critical to creating more engaging and effective learning experiences. This study investigated students’ frustration with academic e-textbooks (e-text frustration) in undergraduate biology classes focusing on the role of cognitive load, motivation to learn, comfort level with e-learning materials, attitudes toward biology, academic preparation, and demographic characteristics in three areas of e-text frustration: (1) frustration with e-text interactions, (2) frustration with technology, and (3) frustration with e-text curriculum. Extraneous cognitive load and motivation were significant predictors of each of the three frustration factors, but the relationships between intrinsic cognitive load, frustration, and motivation varied by an e-text frustration factor.